The Prokaryotic Expression and Bioactivity of the Recombinant Red Fire Ant Venom Allergen Sol i 4

The sting of red imported fire ant （RIFA） could cause serious allergic response in fraction of people. These allergic reactions are mainly caused by its venom, especially venom allergen Sol i 1-4. To produce large amount of RIFA venom allergen Sol i 4 for diagnosis of RIFA allergy and allergen-specific immunotherapy, the gene encoding this protein was amplified and cloned into the prokaryotic expression vector pET43, la. The recombinant plasmid was used to transform competent cells and the recombinant proteins were expressed in E. coll. SDS-PAGE and Western blotting analysis indicated that high-level expression of Sol i 4 protein was successfully achieved. Allergenic activity analysis of the recombinant allergen Sol i 4 was then performed on rabbit. The result showed that the recombinant protein obtained had significant allergenic activity. It indicated that the recombinant allergen Sol i 4 of RIFA venom was successfully expressed in E. coli, which provided foundation for further developing therapeutic and diagnosis reagents of RIFA allergy.     

Cyclic AMP phosphodiesterase in cloned astrocytoma cells: norepinephrine induces a specific enzyme form

The soluble supernatant fraction of homogenates of cloned rat astrocytoma cells (linle C-2A) was subjected to polyacrylamide gel electrophoresis. Two peaks of adenosine 3',5'-monophosphate phosphodiesterase activity were found, corresponding to peaks I and IV of a similarly prepared homogenate of rat brain. Incubating cells with norepinephrine (0.3 mullimolar) caused about a threefold increase in the activity of peak IV but no change in peak I. This increase was completely inhibited by prior inclubation with propranolol (0.1 millimnolar), a beta-adrenergic blocking agent, or with cyclohexamnine (40 micromolar). a protein synthesis inhibitor. Induction of a specific phosphodiesterase form by norepinephrine sitggests another feedback control mechanism whereby an organism can prevent the effects of excessive sympathetic activity.     

Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.     

Serum alpha globulin fraction: survival-and-recovery effect in irradiated mice

An alpha macroglobulin fraction (19S) was isolated from the serum of rats and BC(3)F(1) mice by zonal ultracentrifugation. Both the isologous and heterologous macroglobulin fractions increased survival among BC(3)F(1) mice x-irradiated with 750 roentgens. The mouse macroglobulin fraction also enhanced radiation recovery of hematopoietic tissue as measured by colony-forming assay and iron-59 incorporation into erythropoietic cells. The overall difference in hematopoietic activity in the irradiated (400 roentgens) mice treated with the macroglobulin fraction, in comparison with this activity in the controls, was three- to fivefold in the bone marrow and nine- to tenfold in the spleen between days 4 and 7 after irradiation. This effect was not obtained with the isologous serum protein fraction containing proteins of smaller molecular weight.     

Crystalline fraction I protein: preparation in large yield

Abolut 1 milligram of twice-recrystallized fraction I protein of constant specific ribulose diphosphate carboxylase activity per gram of leaves (fresh weight) has been obtained from each of seven different species of Nicotiana and 14 reciprocal, interspecific F(1) hybrids. Crystals are produced from honmogenates that have only been centrifuged to remove particulate matter.     

苦瓜种子蛋白的双水相提取及抑菌性研究

筛选优化了聚乙二醇(PEG)/硫酸铵双水相的质量分数、分配系数、回收率等影响因素,得到了苦瓜种子蛋白的最佳萃取条件:在26%硫酸铵、22%聚乙二醇条件下,分配系数最小,达0.089,回收率为96%,得到分子量为35.6 kD和12.3kD的苦瓜纯化蛋白。抑菌试验表明,苦瓜种子蛋白对细菌、真菌均有不同程度的抑制作用,且纯化蛋白的抑菌性大于粗提蛋白。     

云南主要地区辣椒分离的黄瓜花叶病毒的亚组鉴定*

 在云南富民、砚山、邱北和南涧等云南主要辣椒产区采集了28个病毒样品,选择应用RT-PCR,Msp I以及EcoR I酶切和克隆测序等分子生物学手段对其中的7种进行检测。经过RT-PCR实验确定其病原为黄瓜花叶病毒（CMV）,同时分离物的Msp I酶切图谱与CMV亚组I的酶切图谱很相似,经EcoR I酶切后也没有出现异常差异。进一步对所检测的黄瓜花叶病毒分离物的外壳蛋白基因进行克隆测序,结果表明,分离物的核苷酸序列与黄瓜花叶病毒亚组I的分离物的核苷酸序列有极高的同源性,达93%~98%; 而与亚组II株系的核苷酸序列同源性仅为77%。因此将所分离到的黄瓜花叶病毒分离物归属于黄瓜花叶病毒亚组I。     

配合饲料对泥鳅鱼体营养成分的影响

研究了 5组泥鳅分别摄食不同配合饲料后鱼体营养成分的变化 .结果表明 ,摄食配合饲料后 ,粗蛋白质( CP)质量分数基本不变 ,粗脂肪 ( EE)质量分数提高 ,含水率下降 ;蛋白质中氨基酸 ( AA)种类齐全 ,蛋氨基( Met)质量分数最低 ,谷氨酸 ( Glu)质量分数最高 .摄食配合饲料后 ,泥鳅鱼体中 AA、必需氨基酸 ( EAA)更丰富 ,EAA/AA更高 ,其中 ,4 #(摄食 4 #配合饲料 ) AA总量、EAA和必需氨基酸指数 ( EAAI)分别是空白组( CK)的 1.56、1.61和 1.0 4倍 ;2 #(摄食 2 #配合饲料 )营养指数是 CK的 1.32倍 .摄食配合饲料后 ,鱼体蛋白质中人体 EAA达到成人需要量模式 ( FAO/WHO/U NU推荐 ) ,基本满足儿童和婴儿的需要 ,同时保持天然泥鳅所固有的风味和品质 .试验组与 CK中 6类呈味 AA质量分数的变异程度 1.752 % ,属可接受范围 .试验组与 CK脂肪酸组成均以 C12 、C16、C18脂肪酸为主 ,此 3系列脂肪酸占总脂肪酸质量分数的 64.7% - 92 .2 % ,其中又以 1#(摄食 1#饲料 )所占比例最高 ,CK最低     

瘤胃微生物不同区系的硝态氮还原、甲烷产生和发酵参数比较

【目的】以硝态氮为惟一氮源,评价体外培养瘤胃微生物不同区系还原硝酸盐的程度以及对甲烷产生和发酵参数的影响。【方法】采用Menke人工瘤胃产气法进行24h厌氧发酵,以硝酸钠、可溶性淀粉和微晶纤维素组成纯合日粮,用物理离心和化学抑制剂相结合的方法,将瘤胃液分成8种区系:全瘤胃液(WRF)、原虫(P)、细菌(B)、真菌(F)、原虫+细菌(P+B)、原虫+真菌(P+F)、细菌+真菌(B+F)和负对照(CTN),测定微生物硝酸盐降解率及发酵参数。【结果】P区系硝酸盐降解率显著高于B区系(P0.001),P+F及B+F区系硝酸盐降解率低;WRF产气量和硝酸盐降解率最高,P+B和P区系次之,这3个区系的甲烷(CH4)、总挥发酸(TVFA)含量和乙酸比例均高于其它区系(P0.001);硝酸盐的还原有利于微生物蛋白的合成,P+B区系的合成量最高。【结论】瘤胃原虫(P)和细菌(B)区系具有高还原硝酸盐(NO3-)和亚硝酸盐(NO2-)能力,原虫区系的硝态氮还原能力更强;真菌(F)区系培养于此发酵底物中,还原硝酸盐的能力很弱,几乎可以忽略。瘤胃原虫和细菌区系是硝态氮还原、产甲烷和微生物蛋白合成的主要区系。     

小蚕人工饲料育对蚕卵生理生化及蚕生长发育的影响

以家蚕原种为研究材料,探讨小蚕用人工饲料育及继代中,蚕生长发育、蚕卵蛋白质质量分数及SOD和CAT活力的变化情况．结果表明：（1）932和芙蓉经过3代的选择,疏毛率分别从14．6％和9．4％提高到52．4％和53．8％,存活率从20．5％、19．2％提高到38．2％、32．0％;而湘晖、7532选择效果不明显．（2）人工饲料育的湘晖和7532的卵蛋白质质量分数都比桑叶育低;而932和芙蓉三代的卵蛋白质质量分数分别保持在52．50～55．00和41．50～52．50mg／g,与桑叶育基本保持不变．（3）蚕卵的SOD和CAT的活力与蚕品种和饲料有关,人工饲料育蚕卵的SOD和CAT活力比桑叶育高．     

Bt Cry1类毒素共性结构域的分析、表达及鉴定

【目的】分析定位Bt Cry1类毒素Cry1Ab、Cry1Ac、Cry1B、Cry1C、Cry1F的共性结构域,克隆并表达共性结构域蛋白,为筛选Bt毒素广谱抗体及建立广谱检测方法打下基础。【方法】利用生物信息学和分子模拟技术,通过SWISS-MODEL同源建模分别对5种Cry1类毒素进行三维建模,并结合Ramachandran plot、ERRAT和Verify3D方法评价模型构象的合理性。通过分析比对5种Cry1类毒素的三维结构,确定DomainⅠ区域作为5种Cry1毒素的共性结构域。以含Cry1Ac基因的苏云金芽孢杆菌库斯塔克亚种为模板设计引物,PCR扩增获得共性结构域DomainⅠ基因,将其经NcoⅠ和NotⅠ双酶切连接至原核表达载体pET-26b(+),构建原核表达载体pET-26b-DomainⅠ。重组质粒经菌液PCR、双酶切以及测序鉴定验证正确后,转化至E.coli BL21(DE3),经终浓度为1 mmol·L~(-1)的IPTG在20℃下诱导表达16h后检测共性结构域蛋白的表达情况。离心收集诱导表达的大肠杆菌菌液,进行超声波破碎处理,收集上清及沉淀,采用SDS-PAGE分析融合蛋白的表达。利用His-Trap HP镍亲和柱纯化上清中的可溶性融合蛋白,经SDS-PAGE电泳、Western blot和ELISA试验验证纯化的共性结构域蛋白的生物活性。【结果】基于氨基酸序列及三维空间比对分析,发现5种Cry1类毒素的DomainⅠ的序列一致性最高,而且它们的DomainⅠ三维结构几乎完全重合,确定DomainⅠ区域作为5种Cry1毒素的共性结构域,通过PCR、双酶切及测序鉴定成功构建原核表达载体pET-26b-DomainⅠ,经IPTG诱导表达、His-Trap HP镍亲和柱纯化获得了可溶性的DomainⅠ共性结构域蛋白,SDS-PAGE和Western blot证实表达的共性结构域蛋白的分子量约为33.4 kD,且能与抗His标签鼠单克隆抗体发生特异性反应,ELISA试验证实共性结构域蛋白与5种Cry1类毒素特异性抗体均具有很强的结合能力,抗原表位分析结果显示共性结构域蛋白具有和完整的Cry蛋白存在多个潜在抗原表位位点的特征,抗原表位区域所占的比例分别为48.4%和63.6%,表明共性结构域蛋白具有良好的免疫原性和免疫反应性。【结论】基于分子模拟与分子克隆技术,成功定位及表达纯化获得共性结构域蛋白,为下一步利用共性结构域为靶标分子制备广谱特异性识别Cry1类毒素抗体打下基础。     

添加菠萝皮对构树叶青贮发酵品质 及蛋白组分的影响

构树叶粗蛋白含量高,富含氨基酸、维生素和微量元素,作为一种非常规蛋白质饲料资源有着良好的应 用前景。为促进构树叶的加工利用,研究了添加菠萝皮对构树叶青贮发酵品质的影响,并利用美国康奈尔净碳水化 合物-蛋白质体系（CNCPS）,对构树叶青贮前后蛋白组分的变化进行了分析。菠萝皮添加量为鲜构树叶的0%、5%、 10%、15%和20% 5个水平,室温青贮60 d。研究结果表明院构树叶缓冲能较高、可溶性碳水化合物（WSC）含量较少, 无添加青贮料其pH 值超过5.0,发酵品质差;添加菠萝皮能显著降低青贮料pH 值、增加乳酸含量,改善青贮发酵品 质;与对照相比,添加20%菠萝皮的青贮料其氨态氮和pH 值含量最低,WSC 含量和乳酸乙酸比最高,无丁酸产生, 青贮发酵品质最好;不添加菠萝皮的构树叶经青贮发酵,约30%的可溶性真蛋白被降解为非蛋白氮,青贮后构树叶 的PA、PC 组分显著增加,而PB3组分显著低于青贮前;菠萝皮原料较构树叶原料含有较高的PA 组分,随着菠萝皮 添加量的增加,各添加组青贮料PA 含量均显著增加。添加菠萝皮虽然能改善构树叶青贮发酵品质,但添加量不宜过 高,否则影响构树叶青贮饲料的营养价值。     

牛IgG轻链的分离纯化及其多克隆抗血清的制备

实验应用凝胶过滤和盐析等方法直接从牛初乳中分离纯化IgG,并应用化学方法断裂回收IgG轻链。分离纯化获得分子量约27.66 ku的IgG轻链蛋白溶液,蛋白质含量为0.105 mg.L-1,浓缩后免疫家兔,获得抗轻链抗血清,效价>11∶6,免疫双扩散和免疫电泳均为特异性条带,表明得到了纯化的IgG轻链及其特异性抗血清。     

棉籽饼粉的理化特性与功能特性

以热榨棉籽饼、冷榨棉籽饼和亚临界萃取棉籽饼为试验材料,对其饼粉的理化特性与功能特性进行研究。结果表明,热榨棉籽饼粉的粗蛋白和游离棉酚质量分数分别为56.23%～60.86%和38～123mg·kg~(-1)。而冷榨和亚临界萃取棉籽饼粉有较高质量分数的蛋白质,未检测出游离棉酚。冷榨和亚临界萃取棉籽饼粉的颜色较浅,亮度较高,分散性好。亚临界萃取棉籽饼粉的吸油能力最强。冷榨和亚临界萃取棉籽饼粉乳化性较优,溶解性较好,而乳化稳定性较差。     

双峰驼诱导排卵活性蛋白氨基酸组成的分析

 应用氨基酸自动分析仪测试了公驼精清及其活性组分。测试结果表明，诱导排卵组分是一种生物活性蛋白质，它富含20种氨基酸；特别是3种碱性氨基酸（赖氨酸、精氨酸、组氨酸）含量达18.1%，明显高于其他活性组分。为测定该活性组分的蛋白质序列，应用数理统计方法计算了该组分多肽分子中的氨基酸残基数以及它的部分氨基酸序列。     

Tuberculin hypersensitivity: studies with radioactive antigen and mononuclear cells

The type and fate of mononuclear cells of guinea pigs hypersensitive to tuberculin were studied by means of purified protein derivative labeled with I(125) and mononuclear cells labeled with tritiated thymidine. Purified protein derivative labeled with I(125) was taken up in vitro by lymphocytes and neutrophils from animals that were either sensitive or nonsensitive to tuberculin, but it was bound more frequently by the cells of sensitive animals. Passive transfer of tuberculin hypersensitivity by means of lymphocytes labeled with tritiated thymidine indicated that significant numbers of radioactive cells migrated to the site where the skin was tested with purified protein derivative only when the test was made immediately after transfusion. Although skin reactions from tests made with purified protein derivative 24 hours after transfusion were comparable to those from tests made immediately, the number of labeled cells at the sites of the later tests was not consistently larger than it was in controls (Histoplasmin reactions). Thus transfused tuberculin-sensitive cells are neither always attracted to the sites of the test with purified protein derivative nor are they required in large numbers at the site for a positive reaction to develop.     

PEG分级法检测绿竹叶片双向电泳中的低丰度蛋白

蛋白质组学研究的关键问题之一是双向电泳(2-DE)中低丰度蛋白的分析,主要是因为相关高丰度蛋白的存在,导致IPG胶条无法吸胀低丰度蛋白,使得低丰度蛋白在2-DE中很难被检测出来。利用梯度浓度的PEG4000沉淀方法来富集绿竹叶片中不同丰度的蛋白质,从而使得低丰度蛋白在凝胶中显现出来。在PEG分级法和传统的全蛋白提取法的2-DE比较中,发现采用PEG分级沉淀法提取的蛋白质的数量以及类别明显增加,高丰度蛋白主要富集在8%和16%的PEG浓度组分中,使得其他组分中的低丰度蛋白与高丰度蛋白组分分别进行2-DE分析。经过对图像和数据的比较、分析,PEG分级法得到的5个组分蛋白点总数超过了1032个,大约是传统蛋白提取方法制备蛋白样品的3倍。